OAC 2018 Nicotinamide Ribose Analysis by LCMSMS

Purpose

Nicotinamide riboside (NR), a pyridine-nucleoside form of Vitamin B3, functions as a precursor to nicotinamide adenine dinucleotide, which is one of the most essential coenzymes in many redox reactions. The FDA granted Generally Recognized as Safe (GRAS) status for NR as a food ingredient in 2016. NR showed instability issues by degrading to nicotinamide, and encapsulation technology is used to stabilize NR. Here, we present a sensitive, fast, and high-throughput quantitative method for NR in microencapsulated products by LC/MS/MS.

Method

Standard and Sample Preparation:  The standard was prepared in water with a concentration range of 50.0-5000 ng/mL. About 20 mg of encapsulated sample was extracted by 10 mL of Heptane, 20 mL of water, and 20 mL of dichloromethane (DCM). The extracted sample was filtered through a 0.20 μm filter before analysis.

UPLC-MS Conditions: UPLC system: Nexera UPLC system including SIL-30AC auto-sampler, controller, column heater, and binary pump (Shimadzu)

Column: 100×2.1 mm, 1.6 μm CORTECS C18 ( Waters)

Mobile Phase A: HFBA and water

Mobile Phase B: Acetonitrile

Flow rate: 0 40 mL/min

Pump Cycle time: 3.5 minutes

MS detector: Triple Quadrupole 5500 MS ( AB Sciex)

Results and Discussion

During method development, the stability of the standard solution was evaluated, and the data indicated that NR is highly sensitive to temperature and basic pH (Fig. 2), ultimately degrading to nicotinamide. To minimize the instability issue, the sample is extracted in amber glassware using a neutral pH diluent at reduced temperature conditions. The extraction methods for the microencapsulated product were also evaluated. To break down the encapsulation shell, an organic solvent is required. Different organic solvents, including hexane, heptane, DCM, chloroform, and acetone, were evaluated, and heptane provided the highest recovery. DCM was also used as an extraction solvent to bring the aqueous layer to the top. The method was successfully developed over the range of 50.0-2,000 ng/mL in microencapsulated product. The specificity experiment showed that there was no significant contribution between analytes/IS and no visible interference peaks showed in blank diluent at the expected retention time (Fig. 3). The LLOQ has sufficient sensitivity (S/N> 10) (Fig. 3). System suitability consisted of six replicate injections of the middle standard solution and was injected before sample analysis and RSD was ≤8.4%. The response revealed that quadratic regression with 1/X weighing factor provides the best, and the correlation coefficient r2 is 0.995 (Fig.4 and Table 2). The accuracy experiment showed that the spiking recovery is within ±20% (Table 3). The %RSD SD 5.63 RSD% of precision was <5.93% (Table 3).

Conclusion

This is the first known published specific, fast, and high-throughput LC/MS/MS assay for the quantification of NR in microencapsulated products.